Nearly 8 million surgical procedures are performed annually in the United States alone to treat tissue and organ dysfunction. Tissue engineering is the development of biological substitutes to restore, maintain, or improve tissue function. Specifically, tissue engineering is a method by which new living tissue are created in the laboratory to replace diseased or traumatized tissue (Langer et al., Science, 260:920-926, 1993).
One particular strategy that has been created to regenerate new tissue is to (i) isolate specific cells from tissue; (ii) expand the isolated cells in vitro; and (iii) implant the expanded cells into the diseased or traumatized tissue so that the implanted cells proliferate in vivo and eventually replace or repair the tissue defect (Langer et al., supra). This technique has been applied to a variety of cell types and tissue defects (for example see Brittberg et al., N. Engl. J. Med., 331:889-895, 1994; Rheinwald et al., Cell, 6:331-344, 1975; Langer et al., supra). Isolated cells could be either differentiated cells from specific tissues or undifferentiated progenitor cells (stem cells). In both cases, establishment of appropriate culture conditions for cell expansion is extremely important in order to maintain or improve their potential to regenerate structural and functional tissue equivalents (Rheinwald et al., supra)
A particular area of focus for the development of tissue regeneration techniques has been correction of defects in cartilagenous tissue. Unlike other tissues, cartilage has little ability to regenerate itself after trauma, disease or as a result of old age. This is due to the avascular nature of normal articular cartilage. Although damage to the superficial chondral plate generally does not heal, the subchondral bone is vascularized, therefore damage to this location does heal to a limited degree. The new cartilage that grows in place of the damaged articular cartilage is called fibrocartilage. Fibrocartilage lacks the durability and more desirable mechanical properties of the original hyaline cartilage. People who suffer joint damage are thereafter predisposed to arthritic degeneration (Freed et al., J. Biomed. Mat. Res., 28:891-99, 1994; Minas et al., Orthopedics, Jun. 20(6):525-538, 1997).
Several different approaches have been taken to repair cartilage tissue. In a method utilizing cartilage explants, cartilage is removed from a body and cultured in vitro for implantation into articular cartilage defects (Sah et al., J. Orthop. Res., Jan. 14 (1):44-52, 1996). Other more current approaches for articular cartilage repair typically consist of harvesting chondrocytes from cartilagenous tissue and seeding the chondrocytes directly onto a three dimensional transplantation matrix material before implantation of the replacement tissue into the damaged area (Freed et al., supra). This technique results in high quality cartilage once regeneration is complete, however this technique requires a large quantity of starting material to be harvested from the patient, resulting in increased patient trauma.
Chondrocytes are isolated from a biopsy, expanded in monolayer cultures until a sufficient number of cells are obtained and implanted into the damaged area of tissue. Implantation requires first, that the cells are either embedded in a gel or associated with a biodegradable polymer scaffold (Brittberg et al., Clin. Orthop., May 326:270-283, 1996; Minas et al., supra; Freed et al., supra; ). The three dimensional nature of these matrices imparts structural integrity to the implant and provides rigid support for growth of the chondrocyte cells into cartilagenous tissue. Although this system has the advantage of requiring fewer cells as starting material, the cartilage obtained by this methods is often of poor quality if the cells are harvested or obtained from skeletally mature donors (adults). Alternatively, progenitor cells from the bone marrow are expanded and used to repair full-thickness defects involving both the articular surface and the underlying subchondral bone (Wakitani et al., J. Bone Joint Surg, 76-A:579-592,1994: Butnariu-Ephrat et al., Clin. Orthop, 330:234-243, 1996).
A distinct challenge presented by this system has been to increase the proliferation rate of the cells during the expansion phase in a manner that results in successful regeneration.
There exists a need for improved expansion techniques for cells that are to be used in tissue engineering.
The present invention pertains to an improved method of expanding cells for use in tissue engineering. It is an aspect of the present invention that expanding cells in the presence of growth factors and hormones stimulates proliferation of the cell population while preserving the properties of the cells necessary for regenerating high quality tissue. It is another aspect of the present invention to provide methods for regenerating tissues with better structural and functional characteristics by recapitulating events occurring during embryonic development. It is yet another aspect of the present invention to provide methods of maintaining or improving the ability of the expanded cells to respond to differentiation stimuli as they regenerate new tissue in vitro or in vivo.
The method of the present invention includes: (i) providing a cell population; (ii) expanding the cell population in the presence of specific biochemical factors; and (iii) using the cells for tissue engineering. A variety of cell types can be used in the present invention. According to the present invention, any cell type desirable for use in tissue engineering that can be isolated is used to regenerate tissue. Non-limiting examples include endothelial cells, muscle cells, chondrocytes and melanocytes. Additionally, any of a variety of biochemical factors that increase proliferation of the cells without losing the quality of the cell can be used in the process of cell expansion. Non-limiting examples of biochemical factors that may be used in the present invention are chondromodulins, platelet derived growth factors, epidermal growth factors, fibroblast growth factor 2, transforming growth factor beta, insulin like growth factors, bone morphogenetic proteins, epidermal growth factor, and platelet derived growth factors.
In a preferred embodiment of the present invention, cartilage tissue for tissue engineering is regenerated using the teachings of the present invention. The present invention demonstrates that chondrocytes isolated from mature cartilage tissue can be expanded in the presence of fibroblast growth factor-2 (FGF-2) and then used to regenerate cartilage tissue. Specifically, FGF-2 added to culture medium during cell expansion helps the cells maintain their potential to regenerate cartilaginous tissue. Specifically, FGF-2 decreases the doubling time of the cell population, while creating a cell population with a homogeneous de-differentiated state and preserving their ability to respond to environmental changes such as responses to growth factors like insulin.
In another preferred embodiment, chondrocytes, preferably mammalian, and more preferably human, are isolated from mature cartilage tissue and expanded in vitro in the presence of fibroblast growth factor 2 (FGF-2) and transforming growth factor betal (TGFxcex2). This expansion allows the de-differentiation of cells while maintaining their full potential for redifferentiation in response to environmental changes.
In another preferred embodiment, the expansion and dedifferentiation of human chondrocytes results in cells that can be redifferentiated into primary chondrocytes for use in tissue engineering. Redifferentiation is performed preferably in a serum-free medium. More preferably, redifferentiation is performed in a serum-free medium containing insulin, transforming growth factor beta (TGFxcex2), and dexamethasone. Most preferably, redifferentiation is performed in a serum-free medium containing insulin, transferrin, selenous acid, linoleic acid, albumin, ascorbic acid, transforming growth factor beta (TGFxcex2), and dexamethasone.
In another preferred embodiment, expansion of cells in the presence of biochemical growth factors for use in tissue engineering also improves the efficiency of tranfection of nucleic acids into the cells. Typically, gene transfer is carried out during monolayer expansion. Therefore, applications where tissue engineering techniques are combined with gene therapy may be utilized in accordance with the teachings of the present invention.
xe2x80x9cDe-differentiationxe2x80x9d: xe2x80x9cDe-differentiationxe2x80x9d is used herein to describe cells that lack differentiated functions and to imply regression to an earlier bipotent or multipotent embryonic state. For example, when chondrocytes from cartilage tissue are released from the cartilage matrix and placed in a monolayer culture, they stop producing characteristic markers that define them as being differentiated. Two such markers for differentiated chondrocyte cells are two well characterized structural macromolecules, cartilage proteoglycan and type II collagen.
xe2x80x9cBioactive moleculexe2x80x9d or xe2x80x9cbiochemical factorxe2x80x9d: xe2x80x9cBioactive moleculexe2x80x9d or xe2x80x9cbiochemical factorxe2x80x9d is used herein to refer to any chemical or protein that can elicit any metabolic response from a cell. A biochemical factor can be a protein, for example a hormone or a growth factor that will stimulate a specific biochemical pathway in the cell. A biochemical factor can also be a simple chemical such as a chemotherapeutic agent. xe2x80x9cBioactive moleculexe2x80x9d is also encompassing of any hydrodynamic factor or signal.
xe2x80x9cExpandxe2x80x9d: xe2x80x9cExpandingxe2x80x9d, xe2x80x9cexpandedxe2x80x9d, or expand is used herein to refer to the process or growing cells in vitro.